Sorbent for resolution of optical isomers and a method for preparation thereof

FIELD: chromatographic sorbents.

SUBSTANCE: invention relates to chromatographic sorbents, which can be used for analysis and preparative purification of optically active compounds. A novel sorbent for resolution of isomers of optically active compounds is developed containing, as chiral selector, macrocyclic glycopeptide antibiotic eremomycin, vancomycin, ristomycin A, teicoplanine, or their aglycons. Method of immobilization of macrocyclic glycopeptide antibiotics is also developed, which resides in that silica gel in aqueous buffer solution is first treated with 3-glycidoxypropyl(trialkoxy)silane and then, in alkaline aqueous or water-organic solution, above-indicated macrocyclic glycopeptide antibiotic is grafted to epoxy group-modified silica gel.

EFFECT: increased selectivity in enantiomer resolution and simplified preparation procedure.

9 cl, 12 dwg, 5 tbl, 9 ex

 

The invention relates to sorbents for chromatography and can be used for analysis and preparative purification of optically active compounds.

Known sorbents for the separation of optically active compounds based on silica with grafted optically active compounds for the separation of enantiomers. Known, for example, sorbent, containing on the surface of silica gel quinine derivatives (SU 1429016, 1988):

Known sorbents containing grafted to the silica group:

or

(SU 1132965, 1132966, 1985).

Known optically active chiral sorbent containing optically active polymer, covalently linked to a solid carrier, the polymer is a net polymer containing optically active derivatives of dicarboxylic acids, diamines, diols or hydroxyacids, preferably derivatives of tartaric acid, and the carrier is an organic or inorganic material. The method of obtaining this sorbent includes securing derivatives of tartaric acid on the surface of the carrier, which is produced by a network of polymerization via hydroxylation in the presence of gedrosian and hydroxylamine and fixed on the carrier in the presence of a catalyst (RU 212139, 1998).

Known sorbent for the separation of racemates of optically active compounds containing media and chiral selector - per-6-amino α-, β, or γ-cyclodextrin or their acetylated counterparts. The method of obtaining this sorbent includes covalent immobilization of chiral selectors on the media, which is carried out by sequential interaction laminirovannogo media with a condensing agent, and then with a reagent selected from groups: per-6-amino-α-cyclodextrin, per-6-amino-β-cyclodextrin, per-6-amino-γ-cyclodextrin, and then with a metal borohydride (EN 2203730, 2003).

However, the known sorbents have enantioselectivity only to certain classes of substances or have insufficient selectivity for the complete separation of the enantiomers.

The closest in technical essence and the achieved result is a sorbent silica gel with chemically grafted glycopeptides antibiotics, such as vancomycin, teicoplanin, teicoplanin aglycone, ristomycin, the structural formula of which is shown in figure 1-4, as well as its production method described in (US 6669842, 2003).

Figure 1 illustreret the structural formula of vancomycin.

Figure 2 illustreret structural formula teicoplanin.

Figure 3 illustreret structural formula aglycone of tampla the ina.

Figure 4 illustreret the structural formula of ristomycin A.

The sorbents exhibit selectivity in the separation of a wide range of enantiomers in water-organic and non-aqueous eluent. However, the enantioselectivity is quite high.

The method of obtaining known sorbents lies in the interaction of the antibiotic with a silicone modifier followed by a chemical binding of the compounds with the surface of the silica gel.

A disadvantage of the known method of producing sorbents with grafted glycopeptides is that the modification of the antibiotic and vaccine derived from it by the silica gel is carried out at a temperature of 90-105°that can lead to the destruction of the molecules of the antibiotic. Uncontrolled changes in the structure of the molecule chiral selector when vaccination can lead to uncontrolled changes in the selectivity of the sorbent. In addition, the disadvantage of this method is the use of absolute organic solvents.

The present invention is to increase the selectivity of the adsorbent in the separation of optical isomers and develop ways to ensure reproducible obtaining selective sorbent and simplify technology.

The problem is solved opisywanym sorbent for XP is matography optical isomers, which contains silica with grafted through the spacer chiral selector - glycopeptides antibiotic, in which the spacer group represented by the formula:

Preferably as a glycopeptides antibiotic it contains eremomycin or its aglycone.

The sorbent may also contain antibiotics selected from the group of: vancomycin, ristomycin a, teicoplanin or their aglycones.

The problem is solved also described a method of producing adsorbent for chromatography of optical isomers, which includes chemical immobilization glycopeptides antibiotic use silicone modifier on the surface of silica gel, which is carried out by first treating the silica gel 3-glycidoxypropyltrimethoxysilane, and then the alkaline aqueous-organic buffer solution of the appropriate antibiotic at a temperature not exceeding 40°C.

Preferably as antibiotic use eremomycin or its aglycone.

You can also use antibiotics, selected from a number of: vancomycin, ristomycin a, teicoplanin or their aglycones.

The following are specific examples of the method.

Example 1.

The example describes a method of immobilization of eremomycin, which is obtained using the strain-producer Amicolatopsis subsp orientalis (EN 211058, 1998). Its structural formula is shown in figure 5.

to 101.1 g of silica gel Kromasil KR100-5-SIL suspended in 500 ml of 0.1 M sodium acetate solution, increased glacial acetic acid to pH 5.5. To the resulting suspension was added 78 ml 3-glycidoxypropyltrimethoxysilane. The reaction mixture was intensively stirred for 2 hours, and then left without heating and stirring for 4 days. Upon completion of the reaction, the modified silica gel was washed with water, ethanol, acetone and filtered. Dried at a temperature of 105°C. According to elemental analysis the carbon content is 5.2%.

The obtained silica gel modified with epoxypropane. Then 1 g of macrocyclic antibiotic - eremomycin dissolved in 15 ml of distilled water. Brought the pH to a value of 8.75, adding dropwise 1 M aqueous solution of KOH. The resulting solution was mixed with 3 g of silica gel with grafted epoxypropane. The resulting reaction mixture was heated at a temperature of 40°With stirring for 14 hours. After the reaction, the sorbent was filtered and washed successively with water, acetonitrile, methanol and diethyl ether. Was dried in a drying Cabinet at 50°C for 4 hours. According to elemental analysis the carbon content is 9.5%.

Thus, the obtained silica with grafted chiral selector:

Silica gel - Si-(CH )3O-CH2-CH(OH)-CH2- Eremomycin

Example 2.

Silica gel modified with epoxypropane obtained as in example 1.

3 g eremomycin dissolved in 25 ml of distilled water. Brought the pH to the value charged 8.52, adding dropwise 1 M aqueous solution of KOH. The resulting solution was mixed with 9.2 g of silica gel with grafted epoxypropane. The resulting reaction mixture was heated at a temperature of 40°With stirring for 14 hours. After the reaction, the sorbent was filtered and washed successively with water, methanol and acetone. Was dried in a drying Cabinet at 50°C for 4 hours. According to elemental analysis the carbon content is 9.5%.

Example 3.

Silica gel modified with epoxypropane obtained as in example 1.

1.2 g of ristomycin And dissolved in a mixture of 15 ml of distilled water and 4 ml of ethanol. Brought the pH to values 8,59, adding dropwise 1 M aqueous solution of KOH. The resulting solution was added to 5 g of silica gel with grafted epoxypropane. The resulting reaction mixture was heated at a temperature of 40°With stirring for 14 hours. After the reaction, the suspension was filtered and washed with water, methanol, acetone. Was dried in a drying Cabinet at 50°C for 4 hours. According to elemental analysis the carbon content is 13.1%.

Example 4.

to 30.1 g silicagel the CSC-G suspended in 150 ml of 0.1 M solution of sodium acetate, increased glacial acetic acid to pH 5.5. To the resulting suspension was added 24 ml of 3-glycidoxypropyltrimethoxysilane. The reaction mixture was intensively stirred for 2 hours, and then left without heating and stirring for 4 days. Upon completion of the reaction, the solid phase was washed with water, ethanol, acetone and filtered. Dried at a temperature of 105°C. According to elemental analysis the carbon content is 4.8%.

1 g of vancomycin dissolved in a mixture of 15 ml of distilled water and 4 ml of ethanol. Brought the pH to a value of 8.50, adding dropwise 1 M aqueous solution of KOH. The resulting solution was added to 5 g of FOC-G-modified epoxypropane. The resulting reaction mixture was heated at a temperature of 40°With stirring for 15 hours. After the reaction, the suspension was filtered and washed with water, methanol, acetone. Was dried in a drying Cabinet at 50°C for 4 hours. According to elemental analysis the carbon content is 8.3%.

Obtained in accordance with the present invention sorbents were tested by separation of optical isomers in the following conditions.

Obtained in examples 1-4 sorbents Packed by suspension polymerization in the column stainless steel size 4,0×250 mm and perform the separation of optical isomers using the method of high performance liquid is ostroy chromatography.

Chromatographic analysis was performed on HPLC chromatograph company KNAUER (Germany) consisting of: pump K-1001, spectrophotometric detector K-2501, thermostat columns Jetstream, with the possibility of temperature control in the range of 5-85°0.1°With manual faucet-spout with a loop of 20 µl. The volume of the sample 5-20 µl. Chromatographic peaks were detected in the range 210-280 nm in accordance with the absorption maxima of the partial connections.

Record the chromatograms, and calculations of the factors holding the split component k, selectivity α and resolution RSwas performed using a hardware-software complex “Multichrome” (the ampersand character, Russia).

As the mobile phase used water and the combined acetonitrile and water-methanol buffer solutions, methanol with the addition of triethylamine and glacial acetic acid. The order of the output L - and D-isomers were determined by standard samples of optically pure compounds.

Example 5.

Table 1 presents the composition of the mobile phases, and in table 2 the results of the chromatographic separation of amino acids and some of their derivatives on the column with sorbent, obtained in example 1, with the grafted eremomycin.

Table 1
Cipher eluentThe composition of the eluent
40 40% of CH3HE IS 60% N2O
60-3,860% of CH3HE is 40% aq. buffer (CH3COOH pH 3.8)
60-4,160% of CH3HE is 40% aq. buffer (1% TEAA pH 4,1)
8585% of CH3HE is 15% aq. buffer (CH3N4, 0,M)
20-4,120% of CH3HE is 80% aq. buffer (0.2% TEAA pH 4,1)
40-4,140% of CH3HE is 60% aq. buffer (0.2% TEAA pH 4,1)
2020% of CH3HE is 80% aq. buffer (NaH2PO4, 0,1M)
42-5842% of CH3SP - 58%H2O

Example 6.

Table 3 presents the results of the chromatographic separation of amino acids and some of their derivatives on the column with sorbent, obtained according to example 3 with the grafted And ristomycin with mobile phase selected from the numbers presented in table 1.

Table 3
ConnectionThe mobile phasek'Lk'DαRS
DL-Trp20-4,1

40-4,1

20
1,81

1,23

2,58
2,85

2,05

of 3.64

1,66

1,41
3,78

3,67

2,99
DL-Phe20-4,1

40-4,1

20
0,67

0,57

0,84
1,34

1,20

1,69
2,00

2,10

2,01
3,69

3,30

2,80
DL-(2-thienyl)-Ala20-4,1

40-4,1

20
0,78

0,73

1,15
1,06

1,07

1,46
1,36

1,46

1,27
2,19

2,70

1,94
DL-Cit20-4,1

20
0,11

0,20
0,18

0,34
1,63

1,70
0,88

1,78
DL-Met20-4,1

40-4,1

20
0,24

0,29

0,32
0,43

0,54

0,60
1,79

1,82

1,87
2,30

2,95

3,31
DL-Ala200,090,333,663,16
DL-Val200,160,583,623,40

Example 7.

In the example presents the results of the separation of atenolol and fenoprofen on the column with sorbent, obtained according to example 4 with immobilized vancomycin.

The results for the separation of enantiomers of atenolol obtained in 100% CH3HE is 0.01% SN3COOH - 0,01% (C2H5)3N and fenoprofen - is the 10% THF - 90% 0.1 M NaH2PO4a pH of 6.5.

The results of the separation are presented in table 4.

Table 4
Connectionk'Lk'DαRS
atenololof 1.341,481,100,95
fenoprofen1,221,321,080,91

Example 8.

Table 5 presents the comparative performance of the sorbents obtained in example 1 and example 3, and sorbent, obtained in a known manner with the grafted And ristomycin (from Ekborg-Ott K.H., Liu Y., Armstrong D.W. // Chirality, 1998, v.10, p.434.). The separation was carried out in eluent identical composition: 50% CH3HE IS 50% H2O.

Table 5
ConnectionColumn with sorbent of example 1Column with sorbent for example 3Column with sorbent, obtained in a known manner
αRSαRSαRS
DL-DOPA4,28there is a 10.03 2,505,111,331,4
DL-Tyrof 5.05to 9.912,664,501,301,52
DL-Trp1,814,302,034,651,231,55
DL-PhGly3,096.42 per3,788,251,231,52
DL-Asn101,532,351,171,56
DL-Met2,234,192,134,221,151,52
DL-α-amino-n-butyric acid3,394,801,301,421,261,56
DL-Cys2,023,45of 1.341,541,181,4
DL-Leu2,54of 3.645,888,301,151,45
DL-Val3,354.09 to3,485,581,231,55
DL-norVal2,653,76is 3.084,801,251,58
DL-Ala2,372,942,563,151,141,45

The results show that the inventive sorbent and the inventive method of immobilization macrocyclic glycopeptides antibiotics on silica gel show higher results on the enantioselectivity than known.

Example 9.

The method described in example 3 was immobilized antibiotic, teicoplanin and aglycones of eremomycin, And ristomycin, vancomycin and teicoplanin. The obtained sorbents showed the ability to separation of optical isomers different compounds.

Chromatogram of the separation of some of the compounds presented in Fig.6-12.

6. The chromatogram of the separation of DL-DOPA on a column with immobilized eremomycin. Eluent: 20% of CH3HE IS 80% 0.1 M NaH2PO4(pH 5.5); 0.7 ml/min Detection: UV 280 nm. Temperature: 22°C.

7. The chromatogram of the separation of DL-Met on a column with immobilized eremomycin. Eluent: 20% of CH3HE IS 80% 0.1 M NaH2PO4; 0.7 ml/min Detection: UV 210 nm. Temperature: 5°C.

Fig. The chromatogram of the separation of DL-His on a column with immobilized eremomycin. Eluent: 20% of CH3HE IS 80% 0.1 M NaH2PO4; 0.7 ml/min Detection: UV 210 nm. Temperature: 22°C.

Fig.9. The chromatogram of the separation of DL-Asp column with immob lisovanim eremomycin. Eluent: 20% of CH3HE is 80% 0,l M NaH2PO4; 0.7 ml/min Detection: UV 210 nm. Temperature: 22°C.

Figure 10. The chromatogram of the separation of DL-PhGly on a column with immobilized ristomycin A. Eluent: 50% CH3HE IS 50% H2O; 0.7 ml/min Detection: UV 220 nm. Temperature: 22°C.

11. The chromatogram of the separation of DL-Asn on a column with immobilized ristomycin A. Eluent: 20% of CH3HE IS 80% 0.1 M NaH2PO4; 0.7 ml/min Detection: UV 210 nm. Temperature: 22°C.

Fig. The chromatogram of the separation of DL-Atenolol on a column with immobilized vancomycin. Eluent: 100% SN3HE IS 0.01% TEA - 0.01% HOAc; 0.7 ml/min Detection: UV 254 nm. Temperature: 22°C.

1. The adsorbent for chromatography optical isomers containing silica with grafted through the spacer chiral selector glycopeptides antibiotic, wherein the spacer represented by a group

2. The sorbent according to claim 1, characterized in that as an antibiotic it contains eremomycin or its aglycone.

3. The sorbent according to claim 1, characterized in that it contains antibiotic And ristomycin or its aglycone.

4. The sorbent according to claim 1, characterized in that it contains antibiotic vancomycin or its aglycone.

5. The sorbent according to claim 1, characterized in that it contains antibiotic, teicoplanin Il is its aglycone.

6. The method of producing adsorbent for chromatography of optical isomers, including chemical immobilization glycopeptides antibiotic use silicone modifier on the surface of silica gel, wherein the first silica gel treated with 3-glycidoxypropyltrimethoxysilane, and then an aqueous or aqueous-organic alkaline buffer solution of the appropriate antibiotic at a temperature not exceeding 40°C.

7. The method according to claim 6, characterized in that the quality of antibiotic use eremomycin or its aglycone.

8. The method according to claim 6, characterized in that the quality of antibiotic use And ristomycin or its aglycone.

9. The method according to claim 6, characterized in that the quality of antibiotic use vancomycin or its aglycone.

10. The method according to claim 6, characterized in that the quality of antibiotic use teicoplanin or its aglycone.



 

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